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Chapter 6
AZD6564, Discovery of a Potent 5-Substituted Isoxazol-3-ol Fibrinolysis Inhibitor and Development of an Enantioselective Large-Scale Route for Its Preparation Staffan Karlsson,1 Daniel Pettersen,2 and Henrik Sörensen*,2 1Early
Chemical Development, Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, SE-431 83 Gothenburg, Sweden 2Medicinal Chemistry, Cardiovascular, Renal and Metabolic Diseases, IMED Biotech Unit, AstraZeneca, SE-431-83 Gothenburg, Sweden *E-mail:
[email protected] We report the discovery and scale up of AZD6564, an oral fibrinolysis inhibitor for treatment of heavy menstrual bleeding. From virtual screening hits, our target compound AZD6564 was discovered by focusing on improving potency, permeability, and selectivity toward GABAa activity. AZD6564 was selected as a candidate for development into clinical studies. The first-generation synthesis of AZD6564 to prepare milligram to gram quantities for in vitro screening relied on a tedious multistep linear synthesis in which chromatography was employed in several steps. Moreover, since no asymmetric approach was identified to install the chiral centers, enantiopure material was obtained after chiral column chromatography. The development of a practical route to the target molecule without recourse to chromatography is described.
Introduction The clotting process, coagulation, is essential in preventing massive blood loss (1). Clotting factors and platelets clump together to form a plug at the injured blood vessel, forming a fibrin clot that stops bleeding and prevents substantial © 2018 American Chemical Society
blood loss. After wound repair, the fibrinolysis mechanism results in dissolution of the fibrin clots to restore normal blood vessel function. Any irregularity in this process can lead to a bleeding disorder. Symptoms of bleeding disorders may include heavy menstrual bleeding (menorrhagia), frequent nosebleeds, excessive bleeding from small cuts, and easy bruising. For example, heavy menstrual bleeding affects 1 in every 5 women; and as such it is one of the most common gynecological complaints (2). There are several medical options available to treat or prevent bleeding disorders and their complications, including iron supplements, nonsteroidal anti-inflammatory drugs (NSAIDs), blood transfusion, contraceptives, and hormonal treatment. One of the most considered treatments of bleeding disorders is the use of fibrinolysis inhibitors, usually represented by ε-aminocaproic acid (EACA) and tranexamic acid (TXA) (Figure 1) (3). They act by blocking the lysine binding site in plasminogen, a key protein in the fibrinolysis mechanism (4). Tranexamic acid (Lysteda©, Cyklo-F©, and Femstrual©) was approved in the United States in 2009 for the treatment of heavy menstrual bleeding, and since 2011 has also been available in the United Kingdom (5). Because its treatment is considered to be cost-effective in many countries, it has been added to the WHO list of essential medicines (6). However, both EACA and TXA suffer from the need of high daily doses (7, 8). High doses of TXA have been reported to lead to gastrointestinal side effects, and there are some rare reports of seizures, hypothesized to be mediated by GABAa activity (9).
Figure 1. Fibrinolysis inhibitors acting by binding to the lysine binding site in plasminogen.
Despite attempts by several companies to find alternative treatments in this area, no other plasminogen lysine binding site inhibitors have reached the market. Therefore, we set out on an internal program to search for novel oral fibrinolysis inhibitors with reduced daily dose and without GABAa-mediated side effects.
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Discovery of AZD6564 TXA, being a relatively small and polar compound with a distinct dipole moment, binds to the lysine binding pocket in the kringle 1 domain of plasmin (10). TXA, a zwitterion, fits the characteristics of the binding pocket very well, as the pocket is quite shallow and heavily polar; thus, it resembles the shape of a typical flashlight battery, with a positive end (piperidine nitrogen) and a negative end (carboxylic acid). Using TXA as the model ligand, we initiated a virtual screen from which the known compound 5-(4-piperidinyl)-1,2-oxazol-3(2H)-one (4-PIOL ) 1 and the pyrazolol 2 were identified as lysine pocket binders (Table 1) (11).
Table 1. Data for Compounds 1 to 3 and Reference Compounds
Key for the interaction of compounds 1 and 2 with the lysine binding site was the positively charged nitrogen in the piperidine interacting with two aspartates (ASP54, 56) and the polar heterocycle’s (i.e., for compound 1 represented by the isoxazol-3-ol moiety) interaction with two arginines (ARG34, 70) (Figure 2).
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Figure 2. Docking overlay of TXA (brighter area) and 4-PIOL 1 (darker area ) in the binding site of plasmin(ogen). Reprinted with permission from reference (10). Copyright 2013 American Chemical Society. Compounds 1 and 2 displayed encouraging potencies, with 1 displaying a 4fold higher potency compared to TXA in a clotlysis assay in human plasma (Table 1). Because of their relatively low molecular weight, they were promising hit molecules from a ligand efficacy (LE) perspective. However, both 1 and 2 suffered from increased activity toward the GABAa receptor compared to TXA (12). In a GABAa binding assay, 1 displayed an IC50 of 35 µM compared to 1600 µM for TXA. Furthermore, both 1 and 2 also suffered from low permeability, as measured in Caco-2 cells (A to B 340:1 cis/trans ratio (Scheme 10). Due to a tight time schedule, we could not evaluate asymmetric hydrogenations, but rather relied on finding a traditional resolution in a downstream step.
Scheme 10. Hydrogenation of 20 to give 21.
Thus, 400 g of pyridine hydrochloride 20 was hydrogenated at ~ 20 bar in methanol with platinum oxide as catalyst. It is important that removal of the spent catalyst takes place under inert conditions as the platinum black formed during the reaction is very pyrophoric. This problem was solved by connecting the bottom outlet of the reactor directly to a homemade filter made from an HPLC column (diameter = 47 mm) with the packing material removed and a Celite® filter cut into shape at the bottom of the column. Analytical samples of the reaction mixture were removed from the top inlet of the reactor. When HPLC-MS and NMR spectra indicated complete reaction, the inlet was closed and the bottom valve to the filter was opened. Pressurizing the reactor with 20 bar of nitrogen forced the mixture through the homemade filter with no back pressure issues to yield a clear filtrate. After line washes with methanol, the reactor and the HPLC filter were then cleaned with water to quench the catalyst. After the water treatment, the removal of the catalyst was a safe procedure.
Eventful Hydrogenations After the trip to our plant in Södertälje, we had 4.4 kg of 20. The factory did not have a working reactor for the hydrogenation and thus the material was transported to our facility in Mölndal for this step. The maximum volume of our dedicated reactor was 5 L, allowing us to run the hydrogenation in three batches of ~ 1.5 kg of 20 in each batch. During our first run at this scale, we used 1.7 mol% of platinum oxide and a starting pressure of 38 bar. Hydrogenation overnight displayed complete consumption of starting material. Thus, the conclusion was drawn that we had probably used an unnecessary excess of catalyst. Consequently, for our second batch on this scale, only 1.05 mol% platinum oxide was used. However, this time hydrogenation overnight still indicated 25% of starting material remained. 166
We decided to add more platinum oxide in order to complete the hydrogenation expediously; that required the addition of the oxide under inert conditions since our reactor contained about 13 g of very fine pyrophoric platinum black. The attempted addition of the finely powdered platinum oxide via a funnel with a back flow of nitrogen resulted in an unfortunate cloud of catalyst geysering above the funnel. Now, accidently 20 bar of nitrogen was blown into the reactor which was virtually full of reaction mixture containing methanol and platinum black. A fair amount of the contents burst out through the addition funnel and sprayed the wall. Instantaneously, the ejected reaction mixture caught fire. The fumes set off the fire alarm and the entire building, with about 200 staff, was evacuated. After the fire brigade had switched off the alarm and the material that remained in the reactor was fully hydrogenated, the third hydrogenation was initiated. But another challenge arose. We were fully aware that as a consequence of the extraordinary diffusion rate of hydrogen and working with a hydrogen pressure of ~ 38 bar, there is no room for imperfections in seals and gaskets. All nuts and bolts had to be tightened carefully and even the tiniest leak would either mean a consumption of hydrogen or a worse scenario. Still, after these precautions, a 3 cm flame was visible standing straight up from the reactor, most likely due to ignition of a minor leak of hydrogen in the presence of platinum black. It was indeed stressful to have an open flame during a hydrogenation operation, but after discussions with our safety officer, we decided that we could tolerate the small flame. Finally, the three batches were combined and after crystallization, the pure (340:1 cis:trans) hydrochloride (82% assay yield) was isolated. Considering the losses due to the fire, we were very pleased with the overall yield.
Resolution of the Piperidine: Enzymatic or Salt Resolution For the reasons previously described, and since very little time was available for identifying an asymmetric route, the chosen route was based on the production of racemic piperidine cis-(±)-21 followed by resolution. Classical and enzymatic resolution approaches were investigated in parallel and evaluated for a large-scale campaign (Scheme 11). For a classical resolution, we screened 21 against an inhouse library of homochiral acids (Figure 3).
Scheme 11. Resolution of cis-(±)-21.
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Figure 3. Commercially available chiral acids used for quick-screen resolution of racemic amines.
Thus, the homochiral acids (0.5–0.7 equiv) were added to the racemic amine in two different solvents (e.g., EtOH and EtOAc), followed by stirring for 24 h. If precipitation occurred, the mixture was filtered and the solid collected was analyzed by chiral HPLC. In this case, it was found that the phosphoric acid 22 gave an almost diastereomerically pure precipitate just after one crystallization. After free-basing/extraction with Na2CO3/EtOAc, (2R,4S)-21 was obtained in >97% ee (as determined by its Mosher amide) and 35% yield (70% of theory). After some experimentation, we found the best conditions to be a 1:2 ratio of methanol:ethyl acetate as solvent. The resolution gave a consistent result also at 200 g scale. However, we were concerned about the accessibility of the chiral phosphoric acid in larger amounts and at a reasonable cost. As a backup plan, we considered the possibility to recycle the phosphoric acid which could be done through extractions. However, we first wanted to await the result from the enzymatic resolution approach. Among all classes of available enzymes, the lipases are known to be straightforward to apply in resolutions. This is especially true of the immobilized 168
enzymes, which can be removed from reaction mixture after completion just through a simple filtration. The immobilized Candida antarctica B enzyme (e.g., Cal B, Novozyme 435) is a well-known lipase that has found wide applicability in various organic transformations. We decided upon using it as a starting point to evaluate the enzymatic resolution of cis-(±)-21. Thus, in a 0.66 M KH2PO4 buffer system and NaOH solution adjusted to pH 8, the substrate was dissolved and Novozyme 435 was added. We were happy to discover that enantioselective hydrolysis did indeed occur; however, luck was not completely on our side since it was the undesired enantiomer that was hydrolyzed. The enantioselectivity, however, was excellent furnishing the unreacted ester (2R,4S)-21 in 34% assay yield and 97% ee. Acid (2S,4R)-23 was removed, together with the immobilized enzyme, by a fairly time-consuming filtration, followed by extraction of our desired ester (2R,4S)-21 using MTBE. The resolution was scaled up and run on 13.8 mol (3.6 kg) scale with similar results. Having established two good approaches for the resolution of (±)-21, we had to choose one of them for further scale up. Both of these approaches were considered good enough to be used on multikilogram scale, but as Novozyme 435 is more readily available at a low cost compared with chiral phosphoric acid 22, it was a straightforward decision in favor of the enzymatic resolution (Scheme 12).
Scheme 12. Resolution of cis-(±)-21 using two different routes. Adapted with permission from reference (23). Copyright 2014 American Chemical Society. Protection of Piperidine (2R,4S)-21 as Carbamate 24 With the establishment of the chiral centers, protection for the amine was next required. As mentioned, protecting groups requiring hydrogenolysis for their removal (e.g., a benzyl carbamate group) were not feasible since 5-substituted isoxazol-3-ols are known to cleave under such conditions (24). Furthermore, 169
we speculated that a larger and more malleable protective group was likely to jeopardize crystallizations of the analog for intermediate (2R,4S)-13, which were important in order to avoid chromatographic purifications. Owing to time constraints, for the initial small deliveries we settled for the established methyl carbamate group as a protecting group (Scheme 13). The carbamate was introduced in quantitative yield using 2-MeTHF, DIPEA, and methyl chloroformate at 0 °C. During an 80 g initial scale up of this step to supply the project with material for the early pre-clinical studies, an incident occurred which is worth some attention. After completing the carbamate formation, (2R,4S)-24 was isolated as a rock-hard solid. Naturally, repeated attempts to dissolve the solid in CH3CN by scraping the inside of the flask with a spatula resulted in an unexpected hole in the flask and the contents ended up in a dirty, hot silicone oil bath. Repeated extractions of the silicone oil with CH3CN selectively extracted our material and in the end gave us back most (> 95%) of the compound in good quality, except for some minor silicone oil. Since the subsequent step was an ester hydrolysis, residual silicone oil was easily removed by extraction. Time constraints required us to keep the methyl carbamate as the protecting group and thus we continued.
Scheme 13. N-protection of piperidine (2R,4S)-21. Hydrolysis of the Ester The subsequent hydrolysis of the methyl ester was a priori considered a simple task. Standard hydrolysis using NaOH or LiOH resulted in 5–20% epimerization of the 4-position of (2R,4S)-24, resulting in acid (2R,4R)-10 as an impurity. To avoid the epimerization, different order of addition of reagents, more dilute conditions, and lower loading of the hydroxides were attempted unsuccessfully. In a previous project, we had experienced the same problem of epimerization during hydrolysis. In that project, in order to minimize epimerization, we developed a new, mild hydrolysis method in which triethylamine was used in combination with lithium salts in wet solvents (30). Probably due to the low basicity of triethylamine compared to alkali hydroxides normally employed in hydrolyses, we could avoid most of the epimerization. Most common solvents could be employed for the hydrolysis, such as CH2Cl2, CH3CN, 2-MeTHF, and, surprisingly, also ester solvents, such as iPrOAc. This gave us the flexibility to choose a solvent which could be used in the previous and subsequent steps, permitting telescoping. By applying the LiBr/Et3N method 170
on ester (2R,4S)-24 in 2-MeTHF, we obtained carboxylic acid (2R,4S)-10 in 99% yield with at most 1% of the trans-isomer (2R,4R)-10 (Scheme 14). Accordingly, by running the protection of (2R,4S)-21 to give carbamate (2R,4S)-24 in 2-MeTHF, it was possible, after an aqueous wash, to use the 2-MeTHF organic layer directly in the hydrolysis step. Using the resulting carboxylic acid (2R,4S)-10 contained in the organic phase in the subsequent β-keto ester formation step was also successful (this will be discussed in the next section). The hydrolysis gave us consistent results also on a larger scale.
Scheme 14. Mild conditions were required in order to maintain the stereochemistry at position 4 in piperidine (2R,4S)-24. Preparation of the β-Keto Ester Next, installation of the β-keto ester was planned as a precursor to building the isoxazole ring. In the first-generation synthesis, this was achieved by pre-activation of the carboxylic acid with carbonyldiimidazole (CDI) followed by reaction with ethyl potassium malonate to give keto ester (2R,4S)-11. For the scale-up, it would be desirable if acid (2R,4S)-10, (which was obtained as a solution in 2-MeTHF), could be telescoped directly to the β-keto ester (2R,4S)-11. The reaction between CDI and acid (2R,4S)-10 to give the intermediate imidazolide was fast and monitored by 1HNMR spectroscopy. Completion was indicated by disappearance of the signal from the starting acid (2R,4S)-10 at 2.63–2.70 ppm. The imidazolide was then treated with a preformed complex of magnesium chloride and ethyl potassium malonate; 30–70% conversion occurred after 2 h. This step was followed by 1HNMR spectroscopy and worked as expected at scales up to 8 g, but caused us a serious headache on going from small lab scale with magnetic stirring to larger scale with overhead stirring. Thus, 80 g of carboxylic acid (2R,4S)-10 was quantitatively converted to the corresponding imidazolide by the straightforward addition of CDI. The resulting suspension was subsequently added to the prestirred mixture of magnesium chloride and ethyl potassium malonate. We were horrified when we did not see any conversion at all after 2 h of reaction time. Heating the mixture was not an option since we were concerned about the stability of both the intermediate imidazolide and the resulting β-keto ester (2R,4S)-11. It was reasoned that we had most likely run into a problem with the potassium magnesium complex. However, we had no idea how to analyze the state of the potassium magnesium complex. 171
Our first thoughts were that we had used poor quality magnesium chloride. After some consideration, a second theory appeared—that the particle surface area of the ingoing magnesium chloride and ethyl potassium malonate was considerably increased by the grinding effect of the stirring bar against the glass walls of the flask used in our small scale reactions. In a reactor equipped with an overhead stirrer, there is quite obviously no grinding. A small scale test was performed with some of the mixture from the reactor mixed with magnesium-malonate complex produced in a flask with a magnetic stirrer. To our relief, our theory was confirmed and a larger portion of the potassium magnesium malonate was prepared in a separate flask with a magnetic stirrer. The resulting suspension/complex was added to the reaction mixture and full progress resulted after overnight reaction. The extra malonate that had been added during our first attempt was removed by washes with aqueous sodium bicarbonate. After this ordeal, keto ester (2R,4S)-11 was isolated in 96% yield as an oil after removal of the solvent (Scheme 15).
Scheme 15. Synthesis of β-keto ester (2R,4S)-11.
As the project progressed onto the next batch, it became apparent that different particle sizes of the magnesium chloride charged gave different outcomes in the reaction. Consequently, the slow conversion was most likely due to low solubility of the magnesium chloride in 2-MeTHF. For the reactor scale batches that followed, we solved the problem by refluxing the magnesium chloride with the ethyl potassium malonate for 5 h in order to make the complex. However, long reaction times were still a serious bottleneck and later batches starting with up to 760 g of carboxylic acid (2R,4S)-10 required as much as five days and additional magnesium chloride/potassium ethyl malonate complex to reach completion. Unfortunately, we could see that epimerization took place during this lengthy procedure and we now had 6% of the wrong epimer at the 4-position in our material. After having finished our final batches (3 kg AZD6564, 5), we discovered that the reaction could be completed in less than 4 h at 25 °C in THF rather than Me-THF, with 12 volumes of solvent instead of 20, and with the same yield/purity outcome. This test reaction gave 76% yield on a 10 g scale. 172
Isoxazol-3-ol Formation The most commonly used route to isoxazol-3-oles commences with the reaction between a β-keto ester and hydroxylamine (Scheme 16) (31). Hydroxylamine may attack the β-keto ester in more than one way, and also equilibrate between the different species formed before cyclization to one of the heterocycles (Scheme 17).
Scheme 16. Formation of the isoxazol-3-ol (2R,4S)-13.
Scheme 17. Simplified mechanistic view. Two isomeric compounds, i.e., the byproduct 2H-isoxazol-5-one (e) and isoxazol-3-ol (f), may result from acid catalyzed cyclization of the possible intermediates formed by reaction between β-keto ester and hydroxylamine. Normally the hydroxamic acid intermediate (b) is not isolated and the reaction mixture is acidified directly. Fast acidification favors formation of desired isoxazol-3-ol (f), while slow acidification has been found to favor formation of the isomeric and undesired 2H-isoxazol-5-one (e). The isomeric structure (e) is frequently formed in alarming amounts even if acidification 173
is fast. A detailed mechanistic study was published in 1986 in which several of the intermediates were identified based on the 13CNMR spectra (32). The formation of the byproduct 2H-isoxazol-5-one (e) during slow acidification has been attributed to equilibration of the hydroxamic acid (b) and the oxime (c) via catalytic amounts of free hydroxylamine. It is well documented that the oxime (c) cleanly gives the 2H-isoxazol-5-one (e) upon acidification. It has also been established that an overly high pH during the reaction between hydroxylamine and β-keto ester (a) will lead to formation of the 2H-isoxazol-5-one byproduct (e) upon acidification (33). It was speculated that at above pH 10 (in aqueous systems), the anion of hydroxylamine (34) will start playing a role in the reaction with the β-keto ester (a). The anion of hydroxylamine may give rise to d. Intermediate d has, to our knowledge, not been positively identified as an intermediate in these reactions but is included in the scheme because its existence and the subsequent reaction path to e was already theorized (32). The mechanisms of these reactions have been studied and discussed in several publications and appear rather complicated (35–37). To make matters worse, early publications in the area contain errors due to confusion of isoxazol-3-ol with the 2H-isoxazol-5-one. Since the synthesis of isoxazol-3-ols from β-keto esters had been extensively studied by several groups, we gathered that we were unlikely to improve the step. Our LO group had used this particular method successfully. Therefore, our keto ester from the previous step was treated with one equivalent of sodium hydroxide in methanol at - 40 °C, followed by one equivalent of hydroxylamine (38). As an extra precaution for avoiding hydroxylamine breakdown and to maintain reproducibility, the reaction was performed in the presence of 0.05 mol% Na2EDTA, which complexes the iron salts. Soluble iron salts are known to be very efficient catalysts (in the ppm range) for the breakdown of hydroxylamine (39). With a heat of reaction of ~ 4 kJ/g, the reaction may be violent at high concentrations. Typical decomposition products are N2, N2O, NO, and NH3. The sampling of the hydroxamic acid formation reaction (2R,4S)-25 was carried out by withdrawing aliquots with a liquid nitrogen cooled pipette followed by immediate HPLC analysis. When conversion of ester (2R,4S)-11 was confirmed (usually after a period of about 4 h at –40 °C), the mixture was added in one portion to 6 M HCl maintained at 80 °C. After a reaction time of 15–30 min, complete consumption of starting material could be confirmed by HPLC-MS. Apart from our desired product, (2R,4S)-13, about 20–30% undesired isomeric isoxazol-5-one, (2R,4S)-12, was also formed as determined by 1HNMR spectroscopy. The mixture was cooled and brought to pH 10.5 with NaOH in order to convert the product to its hydrophilic anion. After removal of lipophilic impurities (among which byproduct (2R,4S)-12 is one) by an extraction with MTBE, the aqueous phase was again acidified with hydrochloric acid to pH 1-2 and extracted with MTBE. Product (2R,4S)-13 was isolated with a purity of 99.9% (by HPLC-MS with detection at 210 nm) in 65–70% yield after removal of the solvent and crystallization from methanol/water.
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Deprotection of Carbamate To Give AZD6564 The final step to produce 5 was the removal of the methyl carbamate protective group from (2R,4S)-13 by treatment with HBr in HOAc (Scheme 18). Our initial experiment at 4.6 g scale first produced material that was brown. Because of the discoloration, the material was purified using preparative HPLC. To our disappointment, the 1HNMR assay was still unsatisfactory and we realized that our compound had passed through the HPLC as the HBr salt. The material was redissolved in pure water and neutralized to pH 6.5 with aqueous ammonia. During the neutralization, the zwitterion precipitated as a crystalline solid monohydrate, which was filterable and gave a 93% assay and a 99.8 area % HPLC purity. The material was identified as a monohydrate, explaining the missing 7% as water. This first deprotection experiment gave a 56% assay yield.
Scheme 18. Removal of the methyl carbamate group to give final product 5 along with byproduct 26. We proceeded with the next 500 g and 280 g starting material campaigns yielding 72% and 68%, respectively, of API. Deprotection of the 500 g material lot displayed a slower reaction rate than the deprotections run on smaller scales in round-bottomed flasks. Most likely, this was an effect of HBr escaping into the head space of the reactor in our large-scale reactions, resulting in a lower concentration of HBr in solution and consequently a slower reaction as compared to the small-scale runs. A small sample of the reaction mixture was removed from the reactor and was subjected to forcing conditions by microwave heating at 120 °C. It was seen that some methylation took place upon this treatment in parallel with the deprotection but it was assumed that the methylation was caused by the high temperature. A short delivery deadline made us take the decision to heat the mixture to a gentle 50 °C, which soon gave rise to serious concerns when the methylation of the API appeared to be about 5%, as determined by HPLC-MS.
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We had grave concerns that the side product would end up in our final material. Our workup at this point consisted of removal of the HOAc/HBr in vacuo followed by dissolution of the residue into 50% aqueous 2-propanol, polish filtration, and precipitation at the isoelectric pH by neutralization with ammonia. To our relief, the precipitated material was 99.9% pure by HPLC-MS and did not contain any detectable methylated material. By extracting the filtrate at pH 14, the pure methylated material was isolated and characterized as byproduct 26 (by NMR analysis) (40). The structure of the byproduct also provided an explanation for its unexpectedly easy removal during workup (Scheme 19).
Scheme 19. Addition of base to the acidic solution of 5 (AZD6564) and 26 precipitates zwitterionic 5. Impurity 26 will stay in solution in its protonated form. The pKa values for 5 have been measured experimentally.
Since the nitrogen in byproduct 26 is N-methylated to give an isoxazol-3-one, tautomerism is impossible and the acidity of the hydroxy group in isoxazol-3-ols will not be displayed. We speculate that the methylation is a simple N-methylation by bromomethane which in turn would be expected to form from the cleavage of the methyl carbamate group. It is thus logical that byproduct 26 does not form a zwitterion and it should consequently not cocrystallize with AZD6564. Our final route for producing up to 500 g of AZD6564 (5) is shown in Scheme 20. The salt resolution using acid 22 was used for producing some of the (2R,4S)-21 and is thus included in the scheme.
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Scheme 20. Summary of the synthesis route for the first 500 g AZD6564, 5. Adapted with permission from reference (23). Copyright 2014 American Chemical Society. 177
GMP Multikilogram Scale Campaigns General Observations in the GMP Campaign Scaling up to the next level (i.e., through production of 3 kg of 5 for phase 1 studies) required starting with ~ 10 kg of 19 and necessitated larger reactors. Therefore, the preparation was transferred to our site in Södertälje. Some small changes were made but the route remained essentially unchanged. The complete route is summarized in Scheme 21.
Scheme 21. GMP route for production of AZD6564 (5). Adapted with permission from reference (23). Copyright 2014 American Chemical Society. During further investigation of the first step in the synthesis, we discovered that PEPPSI-IPr (41) (Figure 4) is a suitable catalyst for the Negishi coupling between 19 and neopentylzinc bromide, requiring only 0.5 mol% of catalyst. The PEPPSI-IPr catalyzed reaction showed a cleaner reaction profile than neopentylmagnesium bromide/Fe(acac)3. Neopentylzinc bromide was also more accessible on larger scale than the equivalent Grignard reagents. 178
Figure 4. Structure of PEPPSI-Ipr. The pyridine coupling partner 19 was not a bottleneck. Starting with 10 kg methyl 2-chloroisonicotinate 19 and 0.5 mol% PEPPSI-IPr in MTBE, followed by the addition of 1.05 equiv of neopentylzinc bromide at < 40 °C, gave an 166 kJ/mol exothermic reaction and a maximal adiabatic temperature rise of 58 °K. After 12 h, the reaction was worked up with citric acid and EDTA. Concentration, acidification with hydrogen chloride in 2-propanol, and precipitation with MTBE gave the hydrochloride salt 20 in 66% yield. For the next step, hydrogenation at a slightly elevated temperature (40 °C) instead of ambient temperature gave cis-(±)-21 hydrochloride in 93% yield, after concentration and precipitation from MTBE. Resolution of cis-(±)-21 was accomplished by enzymatic resolution as in the GLP campaign, but with small alterations. The hydrolysis was simplified by dissolving cis-(±)-21 (13.1 kg) with 1.57 equiv of dipotassium phosphate in 80 L of water and with no further adjustment of pH to give a pH 8 solution. Immobilized Candida antarctica lipase (CALB-T3-150, 3.95 kg) was added, followed by stirring for 40 h at 35 °C. Cooling, addition of 2-MeTHF, and basification with KOH solution gave, after filtration, the desired epimer as its methyl ester retained in the organic phase. The extract was directly diluted with more 2-MeTHF, followed by addition of DIPEA and then methyl chloroformate. The reaction mixture, now containing carbamate (2R,4S)-24, was washed with water followed by addition of triethylamine and LiBr, and heating at 85 °C for 41 h in order to accomplish the hydrolysis to acid (2R,4S)-10. Crystallization was effected after acidic and aqueous workup, concentration, and heptane addition, and gave pure acid (2R,4S)-10 in 31% yield over three telescoped steps (enzymatic resolution, hydrolysis, and carbamate formation). Improvements to the resolution steps were: •
•
For the enzymatic hydrolysis, dipotassium phosphate was used as the single pH regulator in larger excess and without adjustment of pH at any point during the reaction. The enzymatic hydrolysis was carried out at 35 °C instead of 20 °C. 179
•
•
Filtration after the enzymatic step was performed after addition of MTBE and pH adjustment. This removed (2S,4R)-23 by extraction rather than by a slow and tedious filtration. Chiral acid (2R,4S)-10 was crystallized from the concentrated extract by addition of n-heptane.
The resulting acid (2R,4S)-10 was then ready for conversion to keto ester (2R,4S)-11. In a similar manner to the GLP campaign, a 3-fold excess of the magnesium chloride complex with ethyl potassium malonate was prepared under reflux in 2-MeTHF for 6 h. The acid (2R,4S)-10 was activated by adding a solution of the acid to a 1.2-fold excess solution of CDI in 2-MeTHF. By this order of addition, the reaction became more practical as well as consuming less CDI (42, 43).The imidazolide was then added to the previously prepared magnesium chloride complex and the mixture was stirred for 61 h at 25 °C and then for 48 h at 35 °C to give 97% conversion. Acidic workup and assay showed a 77% yield. Improvements in conversion of 10 to 11 were: • •
Reverse order for preparation of imidazolide; acid (2R,4S)-10 was added to CDI in 2-MeTHF. The reaction mixture was heated at 35 °C for 48 h to complete the reaction.
During one of the optimization efforts for the subsequent step, that is, conversion of the β-keto ester (2R,4S)-11 to protected isoxazol-3-ol (2R,4S)-13, the sodium salt of (2R,4S)-25 precipitated and as a result, the reaction failed. We discovered that the reaction could be improved by using triethylamine instead of sodium hydroxide and also running the reaction at –10 °C instead of –40 °C. These operations avoided solid salt formation. Acid treatment in a similar manner as in the GLP route was followed by using MTBE both for workup and crystallization, yielding the isoxazol-3-ol (2R,4S)-13 in 55% yield. Improvements in the conversion of 11 to 13 were: • •
Hydrolysis and reaction with hydroxylamine using triethylamine instead of sodium hydroxide and at –10 °C instead of –40 °C. Crystallization and workup of (2R,4S)-13 with MTBE.
The API formation step was carried out along the lines of the GLP route, with the change that a scrubber with ethylenediamine, sodium thiosulfate, sodium hydroxide, and water was connected to the reactor in order to trap bromomethane, bromine, and hydrogen bromide gases. Thus, carbamate (2R,4S)-13 was heated in 33% HBr/HOAc at 30 °C for 16 h. Evaporation, eventually followed with the addition of water by dissolution in 2-propanol filtration and neutralization using aqueous ammonium hydroxide, gave the crude API. The material was finally stirred in cold water for 20 h to give the final API (5), in 3 kg as a monohydrate in 89% yield and with an ee of 99.9%. The assay of this material was 89.8% and the water content was 10.2%. Table 3 summarizes the first-generation synthesis with the final GMP route. 180
Table 3. Comparison of First-Generation Synthesis with the GMP Route Parameter
First-generation synthesis
GMP route
Number of steps
8
8
Chromatography steps
4
0
Telescoped steps
No
Yes
Chiral column chromatography
Yes
No
0.3%
7%
Yes
No
Difficult
Medium
Overall yield Cryogenic conditions Ease of operation
Conclusions In summary, a first-generation synthesis of the fibrinolysis inhibitor AZD6564, involving four chromatographic purifications and an overall yield of 0.3%, was improved to a chromatography-free synthesis with a 7% overall yield. Two different cross-couplings were demonstrated for the attachment of a neopentyl side chain on pyridine, starting from neopentylmagnesium chloride or neopentylzinc bromide and easily accessible methyl 2-chloroisonicotinate. Hydrogenation of the resulting 2,4-disubstituted pyridine gave rise to racemic 2-(2,2-dimethylpropyl)piperidine-4-carboxylic acid, which was resolved using either chemical or enzymatic means. The enzymatic resolution was selected for our GMP route. After protection of the nitrogen as a methyl carbamate, a particularly mild ester hydrolysis with triethylamine and LiBr was required in the step to follow in order to preserve the stereochemistry. The resulting acid was activated using carbonyldiimidazole and reacted with the preformed complex between magnesium chloride and ethyl potassium malonate to give a β-keto ester. Reaction with hydroxylamine at –10 °C in the presence of triethylamine followed by fast acidification gave a mixture of two isomeric isoxazoles, which were separated by extractions and crystallization. The final step, removal of the methyl carbamate protecting group, gave rise to some N-methylated byproduct, which was easily removed by precipitation and isolation of the end product as its zwitterion.
Acknowledgments A number of chemists have been involved in solving the chemical problems associated with developing the synthesis of AZD6564. In particular, the following people have made important contributions: Leifeng Cheng, Martin Bollmark, 181
Peter Schell, Søren M. Andersen, Robert Berg, Christofer Fredriksson, Catarina Liljeholm, Angéle Cruz, and Fritiof Pontén.
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